Three-dimensional genome organization, in which the linear DNA sequence is partitioned into functionally distinct domains, is essential for the precise deployment of genetic information in every cell type. An increasing number of devastating disorders and diseases have recently been attributed to disruption of genome organization, yet the mechanistic underpinnings remain poorly understood, especially in vivo. In particular, how the activity of binding factors and sequence elements is translated to epigenetic states and three-dimensional genomic domains to govern gene expression remains mysterious. C. elegans provides an experimentally advantageous in vivo model to dissect the mechanisms governing proper establishment of genomic domains. Data in hand points to a novel mechanism in which a sequence-specific factor (SNAP190) acts with components of the RNA polymerase III complex to define a large genomic domain exhibiting tissue-specific, coordinated regulation of thousands of noncoding small RNAs. In this proposal, the powerful genetic and genomic tools of C. elegans will be employed to investigate how SNAP190, RNA polymerase III, cohesin, and other candidate factors, interact with chromatin states (Aim 1), chromosomal looping (Aim 2), and cis- elements (Aim 3) to define the boundaries of, and regulate gene expression within, this unique genomic domain. The successful completion of the proposed experiments will illuminate the poorly understood yet vitally important mechanisms driving large-scale genome organization in vivo.